The transition state and regulation of γ-TuRC-mediated microtubule nucleation revealed by single molecule microscopy

Akanksha Thawani, Michael J. Rale, Nicolas Coudray, Gira Bhabha, Howard A. Stone, Joshua W. Shaevitz, Sabine Petry

Research output: Contribution to journalArticle

Abstract

Determining how microtubules (MTs) are nucleated is essential for understanding how the cytoskeleton assembles. While the MT nucleator, g-tubulin ring complex (g-TuRC) has been identified, precisely how g-TuRC nucleates a MT remains poorly understood. Here, we developed a single molecule assay to directly visualize nucleation of a MT from purified Xenopus laevis g-TuRC. We reveal a high g-/ab-tubulin affinity, which facilitates assembly of a MT from g-TuRC. Whereas spontaneous nucleation requires assembly of 8 ab-tubulins, nucleation from g-TuRC occurs efficiently with a cooperativity of 4 ab-tubulin dimers. This is distinct from pre-assembled MT seeds, where a single dimer is sufficient to initiate growth. A computational model predicts our kinetic measurements and reveals the rate-limiting transition where laterally associated ab-tubulins drive g-TuRC into a closed conformation. NME7, TPX2, and the putative activation domain of CDK5RAP2 do not enhance g-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal g-/ab-tubulin interaction.

Original languageEnglish (US)
Article numbere54253
Pages (from-to)1-34
Number of pages34
JournaleLife
Volume9
DOIs
StatePublished - Jun 2020

All Science Journal Classification (ASJC) codes

  • Neuroscience(all)
  • Immunology and Microbiology(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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